Glycoproteins are abundant in the animal and plant kingdoms as well as in microorganisms. The carbohydrate moieties of glycoproteins have been shown to have multiple functions such as having important roles for cell to cell recognition, cell signalling, protein structure, protein protection, protein stabilisation, and protein to protein interaction. The carbohydrate chains of most glycoproteins are linked to proteins by either N-acetylglucosamine (GlcNAc) bound to the free amino groups of asparagine residues (N-linked carbohydrate) or by N-acetylgalactoseamine (GalNAc) bound to the hydroxyl groups of serine or threonine residues (O-linked carbohydrate). The carbohydrate moiety at each site is often composed of several monosaccharide units forming complex structures. Most glycoproteins contain several different carbohydrate chains in one molecule. The structures of asparagine-linked carbohydrates are of three types; high-mannose, complex and hybrid type. These all have a common pentasaccharide core, Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc. In high-mannose type structures further mannose units are coupled to this core and in complex type structures, fucose, galactose, N-acetylglucoseamine and sialic acid are commonly found. Two or more antennae of monosaccharide units might extend from the common core. The hybride type of carbohydrate chains contains antennae of both the high mannose type and complex type.
Hemophilia is a group of hereditary genetic disorders that impair the body's ability to control blood clotting or coagulation. In its most common form, Hemophilia A, clotting factor VIII is deficient. In Hemophilia B, factor IX is deficient. Hemophilia A occurs in about 1 in 5,000-10,000 male births, while Hemophilia B occurs at about 1 in about 20,000-34,000. The Factor VIII protein is an essential cofactor in blood coagulation where factor IXa converts factor X to its activated form. The deficiency of factor VIII and factor IX, respectively, can be treated with plasma-derived concentrates of factor VIII and factor IX or with recombinantly produced factor VIII and factor IX. The treatment with factor VIII or factor IX concentrates has led to a normalized life of the hemophilia patients. However, a certain percentage of the Hemophilia A patients develop inhibitory antibodies directed against the infused factor VIII product resulting in an impaired therapeutic effect. In recent years the recombinant products produced in hamster cell lines have become more frequently used. However, some reports might suggest that these products cause a higher incidence of inhibitor formation than the plasma-derived ones (Ettingshausen and Kreuz, 2006; Hay, 2006).
The nascent form of factor VIII is a single chain of about 300 kDa consisting of domains described as A1-A2-B-A3-C1-C2 (Gitschier et al., 1984; Toole et al., 1984; Vehar et al., 1984; Wood et al., 1984). The protein undergoes processing prior to secretion into blood resulting in a heavy chain of about 200 kDa consisting of the A1-A2 domains and the major part of the B-domain and a light chain of 80 kDa consisting of the A3-C1-C2 domains (here called full-length factor VIII). The B-domain has been shown to be dispensible for the coagulant activity of factor VIII (Andersson et al., 1986; Brinkhous et al., 1985; Pittman et al., 1993). Therefore, it is possible to use factor VIII with a deleted B-domain in therapeutic use for the treatment of hemophilia A (Courter and Bedrosian, 2001).
In blood circulation the intact factor VIII is present in a tight complex with the high molecular mass protein, von Willebrand factor (vWF). vWF is not an enzyme and therefore has no catalytic activity. Its primary function is binding to other proteins, particularly factor VIII and it is important in platelet adhesion to wound sites. vWF is a large protein consisting of multiple monomers with a total molecular mass up to 20.000 kDa. As a carrier protein of factor VIII in the blood circulation vWF protects it from proteolytic degradation and thereby increases the in vivo survival of factor VIII. Furthermore, it has been suggested that the binding of vWF to factor VIII has a protective effect against recognition of factor VIII by the immune system and thereby resulting in a decreased risk for development of inhibiting antibodies against factor VIII (Gensana et al., 2001; Kailas and Talpsep, 2001).
The full-length factor VIII is a heavily glycosylated protein with 25 potential sites for N-linked glycosylation, of which the majority reside in the B-domain. Only six of these sites are localized within the A and C domains. Previously published data showed that four of the potential sites in the factor VIII A and C domains are glycosylated. These were Asn 41 and Asn 239 in the heavy chain and Asn 1810 and Asn 2118 in the light chain (Lenting et al., 1998; Sandberg et al., 2001). Thirteen sites for O-linked glycosylation were found in full-length recombinant factor VIII, most of them located in the B-domain (Lenting et al., 1998). The B-domain-deleted factor VIII, ReFacto®, was reported to have two sites substituted with O-linked carbohydrate chains (Sandberg et al., 2001).
Recombinant human proteins are most often expressed in murine cell lines. This means that although a human gene construct has been used for expression the proteins have murine glycosylation patterns. One such example is the antigenic carbohydrate group unbranched Galα1-3Gal, present in recombinant proteins produced from these cell lines (Galili et al., 1985; Hokke et al., 1995). However, this carbohydrate group is not present in native human glycoproteins. Another example is the composition of sialic acid types present. Recombinant proteins derived from murine cell lines have been reported in addition to the major form of sialic acid, N-acetylneuraminic acid (Neu5Ac), to also contain some percentage of the antigenic sialic acid, N-Glycolylneuraminic acid (Neu5Gc) (Hokke et al., 1990). Neu5Gc is normally absent in native human proteins due to a mutation in the gene coding the enzyme CMP-N-acetyl neuraminic acid hydroxylase, but present in all other mammalian glycoproteins (Chou et al., 2002). However, Neu5Gc has been reported to be present in rapidly growing cells such as human cancer cells and in human embryonic cells (Kawashima et al., 1993; Marquina et al., 1996; Tangvoranuntakul et al., 2003).
Most humans have been shown to have circulating antibodies both against the Galα1-3Gal epitope and the Neu5Gc.
Today, the recombinant human factor VIII products for therapeutic use are all derived from murine cell lines, which mean that they have a murine glycosylation pattern. Thus the antigenic carbohydrate group, unbranched Galα1-3Gal, might be present in factor VIII produced from these cell lines (Hironaka et al., 1993). However, this carbohydrate group is not present in the plasma-derived human factor VIII. Another example is the presence of the antigenic sialic acid N-glycolylneuraminic acid in factor VIII from murine cell lines.
Methods to produce recombinant factor VIII protein in human cell lines have been disclosed in the prior art. US-A-2006/099685 relates to a process for the production of a recombinant factor VIII protein in human embryonic retina cells focusing on the importance of said cells to express at least one adenoviral E1A protein. The recombinant protein produced according to this procedure is said to have a human glycosylation pattern different from the isolated human counterpart.
WO-A-2007/003582 provides a method for transfection and production of human recombinant proteins, in particular blood proteins, in human cell lines under serum- and protein-free conditions, stably transfected with a specific vector carrying the gene coding for the protein of interest.
US-A-2003/0077752 relates to glycosylated proteins having human factor VIII activity. The glycosylation pattern of this product contains alpha-(2,6)-linked sialic acid and bisecting N-acetylglucosamine linked to a core beta-mannose.
EP-A-0774261 discloses complexes of factor VIII and vWf and their therapeutic value.
B. Mei in Molecular Biotechnology Volume 34, 2006, 165-178; V. Picanco-Castro et al. in Genetics and Molecular Research 7 (2):314-325 (2008); M.-H. Rodriguez, British Journal of Haematology, 127, 568-575; G.-Ch. Gil, Glycobiology vol. 18 no. 7 pp. 526-539 as well as WO-A-2008/092643 disclose the production of recombinant factor IX or factor VIII in human cells.
It would be desirable with a recombinant human factor VIII product and recombinant human factor IX product with a human glycosylation pattern in order to minimise the immunogenicity of the recombinant protein when used in therapy.